Immunohistochemical identification and assessment of the location of leptin, visfatin and chemerin in the liver of men with different body mass index.

BACKGROUND: Adipokines such as leptin, visfatin and chemerin play a pivotal role not only in the pathogenesis of excessive weight gain but also impact on hepatic metabolism. However, alterations in the production of these peptides in the liver of overweight individuals have not been fully elucidated yet. The aim of the study was to evaluate changes in leptin, visfatin and chemerin biosynthesis in the liver of men with different BMI. METHODS: Fourteen adult men without symptoms from the digestive system were recruited. Research material consisted of liver samples. Study participants were divided into two groups: lean (BMI ≤ 25 kg/m2) and overweight subjects (BMI > 25 kg/m2). Paraffin liver sections were processed by immunohistochemistry for detection of leptin, visfatin and chemerin. Hepatic expression of leptin, visfatin and chemerin genes was determined by qRT-PCR method. RESULTS: Increased immunoreactivity for leptin and chemerin, and decreased immunoreaction for visfatin were observed in the liver of overweight men in comparison to lean subjects. Overweight subjects with hepatic steatosis displayed increased immunoreactivity for leptin and weaker immunoreaction against visfatin and chemerin in the liver, compared to individuals with normal organ structure. Expression of leptin and chemerin was enhanced in the liver of overweight individuals, with the highest expression observed in subjects with hepatic steatosis. Conversely, expression of visfatin in the male liver was decreased in overweight subjects and those with and liver steatosis. CONCLUSIONS: The present study proves that the expression of leptin, visfatin and chemerin in the male liver is altered in overweight individuals. Our report also indicates the potential importance of these peptides in hepatic steatosis associated with overweight.

Altered cannabinoid receptor expression in pancreatic islets in experimental model of uraemia.

BACKGROUND: Uraemia leads to a number of metabolic and hormonal disorders including defective carbohydrate metabolism. Endocannabinoids exert their effect on insulin and glucagon secretion via activation of specific receptors named CB1 and CB2. For this reason and the absence of reports on location and immunoreactivity of CB1, CB2 receptors compared to immunoreactivity of insulin- and glucagon-secreting cells in experimental uraemia, the author decided to investigate this issue. The aim of the present study was the immunohistochemical localisation and evaluation of cannabinoid receptors (CB1, CB2), insulin and glucagon in the pancreatic islets of uraemic rats. MATERIALS AND METHODS: Fragments of the rat's pancreas were collected 28 days after surgical resection of one kidney and removal of 70% of the other kidney cortex. Paraffin-embedded sections were stained with haematoxylin-eosin and immunohistochemical reactions were performed with the use of a specific antibody against CB1-, CB2-receptors, insulin and glucagon. RESULTS: It was revealed the decreased immunoreactivity of the CB1 receptor and higher intensity of the immunohistochemical reaction against CB2 receptor as compared to the value in the control animals. Significantly higher immunoreactivity of glucagon-positive cells and weaker immunoreactivity of insulin-positive cells were observed in pancreatic islets of uraemic rats. CONCLUSIONS: The obtained results indicate the involvement of cannabinoid receptors in the pathomechanism of carbohydrate metabolism disorders, associated with abnormal secretion of hormones by the α and ß cells in uraemia.

Decreased immunoreactivity of visfatin in the pancreas and liver of rats with renovascular hypertension.

Hypertension is one of the major endocrine and metabolic disorders, in which visfatin plays a significant role. The objective of this study was to evaluate the immunoreactivity of visfatin in pancreas and liver of “two kidney, one clip” (2K1C) renovascular hypertension model in rats. The studies were carried out on the pancreas and liver of rats. After a 6-week period of the renal artery clipping procedure, 2K1C rats developed a stable hypertension. Paraffin sections were stained with hematoxylin and eosin (for general histological examination) and processed for immunolocalization of visfatin. The intensity of immunohistochemical reaction was measured using Nikon NIS-Elements Advanced Research software. The hypertension significantly weakened the immunohistochemical reaction exhibiting visfatin in the pancreas and liver of hypertensive rats, compared to control animals. The changes induced by hypertension in the visfatin-containing cells in the pancreas and liver of the rats are discussed and needs further study.

Quantitative evaluation of CART-containing cells in urinary bladder of rats with renovascular hypertension.

Recent biological advances make it possible to discover new peptides associated with hypertension. The cocaine- and amphetamine-regulated transcript (CART) is a known factor in appetite and feeding behaviour. Various lines of evidence suggest that this peptide participates not only in control of feeding behaviour but also in the regulation of the cardiovascular and sympathetic systems and blood pressure. The role of CART in blood pressure regulation led us to undertake a study aimed at analysing quantitative changes in CART-containing cells in urinary bladders (UB) of rats with renovascular hypertension. We used the Goldblatt model of arterial hypertension (two-kidney, one clip) to evaluate quantitative changes. This model provides researchers with a commonly used tool to analyse the renin-angiotensin system of blood pressure control and, eventually, to develop drugs for the treatment of chronic hypertension. The study was performed on sections of urinary bladders of rats after 3-, 14-, 28-, 42 and 91 days from hypertension induction. Immunohistochemical identification of CART cells was performed on paraffin for the UBs of all the study animals. CART was detected in the endocrine cells, especially numerous in the submucosa and muscularis layers, with a few found in the transitional epithelium and only occasionally in serosa. Hypertension significantly increased the number of CART-positive cells in the rat UBs. After 3 and 42 days following the procedure, statistically significantly higher numbers of CART-positive cells were identified in comparison with the control animals. The differences between the hypertensive rats and the control animals concerned not only the number density of CART-immunoreactive cells but also their localization. After a 6-week period, each of the rats subjected to the renal artery clipping procedure developed stable hypertension. CART appeared in numerous transitional epithelium cells. As this study provides novel findings, the question appears about the type of connection between hypertension and the functioning and activity of CART in the urinary tract (UT). The study gives rise to the assumption that high blood pressure can be a factor that intensifies CART secretion. In conclusion, the endocrine system of the urinary tract is modified by renovascular hypertension. This may affect the production of hormones and biologically active substances and contribute to the development of possible hypertension complications. In order to fully comprehend the role of the CART peptide in blood pressure regulation, further analyses are necessary.

The skin remodeling in type 1 diabetes and insulin resistance animal models.

The skin matrix metalloproteinase 3, tissue inhibitors of matrix metalloproteinase 2 and collagen III content changes in type 1 diabetes and insulin resistance treated with insulin and metformin were studied. Healthy adult male Wistar rats were obtained from experimental animal house, Department of Experimental Pharmacology, Medical University in Bialystok. The rats were divided randomly into five groups of 8 rats each. Control rats were injected intraperitoneally by NaCl. Type IDDM was induced by a single injection of Streptozocin. Insulin resistance was induced by a high-fat diet. The chosen groups of rats were also treated with insulin or metformin. ELISA Kits (USCN Life Science, China) were used to measure content of matrix metallo-proteinase 3 (ELISA Kit for Matrix Metalloproteinase 3 - MMP3), tissue inhibitor of matrix metalloproteinase 2 (ELISA Kit for Tissue Inhibitors of Metalloproteinase 2 - TIMP2) and content of collagen type 3 (ELISA Kit for Collagen Type III - COL3). The results were reported as a median. The statistical significance was defined as p<0.05. Type 1 diabetes and insulin resistance have significantly reduced the quality of the skin, shown by the increase in content of matrix metalloproteinase 3 and the decrease in content of tissue inhibitors of matrix metalloproteinase 2. Type 1 diabetes and insulin resistance have reduced the quality of the skin expressed by type III collagen content decrease but for future studies it is recommend to determine rat interstitial collagenase, MMP-13, as well. Insulin and metformin treatment improved the quality of the diabetic skin, demonstrated by the type III collagen content increase.

Evaluation of CART-, glucagon-, and insulin-immunoreactive cells in the pancreas of an experimental rat model of unilateral renal artery stenosis.

Hypertension is one of the most frequently occurring diseases worldwide. Approximately 10% of the population with hypertension reveal the secondary type of hypertension. The aim of this study was to evaluate the cells containing CART, insulin and glucagon in the pancreas of rats with renovascular hypertension. An experimental model of hypertension in rats according to Goldblatt (2K1C model of hypertension) was used in the study. The experimental material (pancreas) was collected in the 6th week of the study. Cells containing CART, insulin and glucagon were evaluated using immunohistochemical and morphometric methods. Pancreatic islet cells were evaluated based on the number and intensity of staining. The investigation showed an increase in the number and immunoreactivity of CART containing cells, 6 weeks after partial unilateral ligation of the renal artery. There was a significant decrease in the number of glucagon-IR cells. Although intensity of staining these cells did not change. No differences were observed in the number and staining affinity of insulin-containing cells. On the basis of the study it can be stated that the endocrine system of pancreas undergoes changes in the course of renovascular hypertension. This may affect the production of hormones and contribute to the development of possible hypertension complications.

Quantitative and qualitative evaluation of CART-containing cells in adrenal glands of male rats with hypertension.

Adrenal activity is stimulated and secretion of stress hormones is increased during advanced stages of renovascular hypertension. The literature suggests that the neuropeptide, cocaine and amphetamine regulated transcript (CART), might regulate adrenal secretory function and thus could influence its activity. We assessed potential quantitative and qualitative changes in the cells that contained CART in the adrenal glands of rats with renovascular hypertension. The renal arteries of ten rats were subjected to a clipping procedure, i.e., two-kidney one-clip (2K1C) model of arterial hypertension, and after 6 weeks each rat developed stable hypertension. CART was localized using immunohistochemistry. CART was detected in a large population of cells in the medulla, sparse nerve fibers in the cortex and the capsule of the adrenal gland. The population of CART-positive cells in adrenal glands of two kidney-one clip (2K1C) treated rats was greater and their immunoreactivity was increased compared to controls. Similarly, the length, width, area and diameter of CART-immunoreactive cells were significantly greater in the hypertensive rats than in controls. We demonstrated that renovascular hypertension alters the number and immunoreactivity of CART-containing cells in adrenal glands.

S100A6 and its extracellular targets in Wharton's jelly of healthy and preeclamptic patients.

INTRODUCTION: In this work we compared the level, localization and binding partners of a calcium binding protein, S100A6, in extracellular matrix of Wharton's jelly of healthy and preeclamptic patients. METHODS: Studies were performed on the umbilical cords taken from 10 newborns delivered by healthy and 10 newborns delivered by preeclamptic mothers. To characterize S100A6 in Wharton's jelly immunoblotting and immunohistochemistry were applied. For identification of S100A6 targets pull down assays and mass spectrometry were performed. Direct interaction of S100A6 with its targets was checked by ELISA while co-localization of these proteins was analyzed by immunofluorescence staining. RESULTS: We have found that the level of S100A6 in Wharton's jelly is higher in patients with preeclampsia than in healthy ones and that post-translational modifications of S100A6 in preeclamptic tissue are different than those of S100A6 in control. We have identified several proteins that might interact with S100A6, among them are lumican and PRELP, found in Wharton's jelly of healthy and preeclamptic patients, and IGFBP-1 identified, as an S100A6 target, only in preeclamptic tissue. We have shown that the interactions between S100A6 and these proteins are direct and that IGF-1 competes with S100A6 for binding to IGFBP-1. CONCLUSION: In Wharton's jelly of preeclamptic tissue S100A6 is up-regulated and binds to different targets than in control. This suggests involvement of S100A6 in development of preeclampsia.

Distribution of cocaine and amphetamine regulated transcript in ureters and urinary bladder of hypertensive rats.

Cocaine and amphetamine regulated transcript (CART), a neuropeptide of the central and peripheral nervous system plays an essential role in maintaining body homeostasis by regulating body temperature, orexia, digestive motility and blood pressure. Very few studies describe the relationship of hyperten¬sion with CART. Therefore, the present research was undertaken to identify, locate and determine the number of CART-immunopositive neuroendocrine cells (NE) and structures in the urinary bladder and ureter of rats with experimentally induced nephrogenic hypertension. The experiments were conducted on 20 Wistar rats in which hypertension was experimentally induced by applying a clamp on the left renal artery based on the two kidney, one clip experimental model (2K1C). After 6 weeks, fragments of the ureters and urinary bladder were sampled from rats with permanent hypertension. Immunohisto¬chemical analyses revealed a salient effect of renovascular hypertension on the neuroendocrine system of rat ureters and urinary bladder. Differences in the number of neuroendocrine cells and in the density of CART-positive structures were identified between the hypertensive and normotensive (control) rats. Hypertension greatly increased the number of NE cells and the density of CART- immunoreactive (IR) structures in the analysed urinary system organs.

Comparative evaluation of gastric ghrelin cells and levels of hormone in the serum of healthy women and men.

Due to difficulties in obtaining human material, most of the data concerning the site of occurrence and synthesis of ghrelin are based on animal studies. There are only few reports describing ghrelin-containing cells in the human digestive tract, based on the limited human material obtained during surgery or biopsy. The aim of this study was to compare and evaluate the distribution and morphology of ghrelin cells in the stomach and the levels of hormone in the serum of healthy men and women. The study included 18 subjects with normal gastric mucosa (12 men and 6 women). Immunohistochemistry was performed using rabbit anti-ghrelin (human) antiserum. Ghrelin level in serum was measured by ELISA. The total number of ghrelin positive cells was greater in the stomach of women than men. Ghrelin-immunoreactive cells were more elongated and larger in the stomach of women. The serum ghrelin level was higher in men than in women. Ghrelin concentration in serum correlates negatively with body mass index and weight in both genders, whereas the correlation between ghrelin level and age was positive in women and negative in men. The number of cells containing ghrelin in the stomach does not reflect the serum hormone levels. The differences in gastric ghrelin cells and ghrelin levels in serum between women and men, indicate that secretion of hormone can be under control sex hormones or other unknown factors.

Cocaine- and amphetamine-regulated transcript : identification and distribution in human gastrointestinal tract.

Cocaine- and amphetamine-regulated transcript (CART) was identified in the central and peripheral nervous system, including the gastrointestinal tract of rodents and pig. CART was also expressed in neuroendocrine cells of the rats stomach antral mucosa. The knowledge of the presence and functional role of CART peptide in the human alimentary tract is very limited due to difficulties in obtaining human samples (especially from healthy individuals). The presence of CART peptide in the gastrointestinal tract of the human was investigated immunohistochemically. CART-immunoreactive (IR) neural structures were observed in all studied fragments of alimentary tract. CART-like immunoreactive nerve fibers were numerous within the muscle in layers of muscularis externa and in the myenteric plexus of all gastrointestinal segments (from esophagus to colon), while they were moderate or few in density in other layers of gastrointestinal tract. The presence of CART peptides in the neuroendocrine cells was demonstrated predominantly in the pyloric, duodenum and fundus, and only few in the rest parts of the small intestine. CART-IR neuroendocrine cells could not be detected in the mucosa of large intestine. The present study reports for the first time a detailed description of the CART distribution pattern within the human alimentary tract. Our findings may hopefully provide some contribution towards a more complete and comprehensive understanding of the function and role of the CART peptide in the alimentary system.

Dynamics of calcitonin gene-related peptide-like cells changes in the lungs of two-kidney, one-clip rats.

Taking into consideration renal hypertension-induced homeostatic disorders and the key role of calcitonin gene-related peptide (CGRP) in many, systemic functions regulating systems, a question arises as to what an extent arterial hypertension affects the morphology and dynamics of pulmonary CGRP-immunopositive cell changes. The aim of the present study was to examine the distribution, morphology and dynamics of changes of CGRP-containing cells in the lungs of rats in the two-kidney, one-clip (2K1C) renovascular hypertension model. The studies were carried out on the lungs of rats after 3, 14, 28, 42, and 91 days long period from the renal artery clipping procedure. In order to identify neuroendocrine cells, immunohistochemical reaction was performed with the use of a specific antibody against CGRP. It was revealed that renovascular hypertension caused changes in the neuroendocrine, CGRP-containing cells in the lungs of rats. The changes, observed in the neuroendocrine cells, depended on time periods from experimentally induced hypertension. The highest intensity of changes in the neuroendocrine cells was observed in the lungs of rats after 14 days from the surgery.

Immunohistochemical identification and localisation of gastrin and somatostatin in endocrine cells of human pyloric gastric mucosa.

The detailed description of the distribution of endocrine cells G and D producing important hormones that regulate activation of other cells in the human stomach may be a valuable source of information for opinions about mucosa changes in different diseases of the alimentary tract. The density and distribution of immunoreactive G and D cells in the pylorus of humans (donors of organs) were evaluated. The pylorus samples were collected after other organs were harvested for transplantation. The number of G cells in the pyloric mucosa of healthy people was higher than the number of D cells. G and D cells were distributed between columnar cells of epithelium mucosa. Multiform endocrine cells generally occurred: gastrin in the middle third of the mucosa and somatostatin cells in the basal half of the pyloric mucosa. The investigation of the pyloric part of the healthy human stomach showed a characteristic distribution of cells that reacted with antisera against gastrin and somatostatin.

Removal of dental amalgam fillings and its influence on saliva morphological picture - case report.

The influence of dental restorative materials on patients' general and oral health is the main interest of many researchers but the question of their safety is still under consideration. An otherwise healthy 23-year-old patient with no history of oral abnormalities was examined. Dental amalgam restorations were replaced by composite resin material. Salivary smears prepared two days and two weeks after the amalgam removal were compared with those taken before the procedure.

The ghrelin-positive cells number is increased in duodenum in children with celiac disease.

Ghrelin is predominately produced in the stomach, but new findings indicate that the intestinal wall is an important source of the hormone. In patients with shortbowel syndrome, reduction in the intestinal tissue resulted in a decrease in the circulating ghrelin levels. Since in celiac disease (CD) intestinal mucosa atrophy is the main finding, alterations in duodenal ghrelin-positive cell population can be expected. The aim of the study was to evaluate the density of ghrelin-positive cells in the duodenum of CD children and its relationship with body mass index (BMI) and clinical presentation. The study included 31 consecutive patients with newly diagnosed CD [BMI SD scores (BMISDS) -0.926+/-1.496]. The control group consisted of 21 children (BMISDS -0.517+/-1.186], diagnosed with growth retardation, anemia or abdominal pain. All the patients underwent endoscopy with biopsy samples taken from distal duodenum. Immunohistochemistry was performed using rabbit anti- ghrelin (human) antiserum. The number of ghrelin-positive cells in the duodenum was significantly higher in children with CD than in controls (14.82+/-11.12 vs 5.69+/-5.02, p<0.0013). The density of ghrelin-positive cells in the duodenum did not correlate with age, pubertal status, BMISDS or clinical presentation. In the duodenum of CD children, the number of ghrelin-positive cells is increased compared with the control patients. The population of ghrelin-positive cells in the duodenum does not simply reflect an altered mucosal morphology or failure to thrive but is under the influence of other conditions.

Quantitative distribution and localization of calcitonin gene-related peptide-like cells in the stomach of two kidney, one clip rats.

The majority of research for the calcitonin gene-related peptide (CGRP) in the stomach in the hypertension has been devoted to the submucosal blood flow, and no attention has been paid to its quantitative distribution in the gastric neuroendocrine cells. The aim of the present study was to examine the number and distribution of CGRP-containing cells in the pylorus of "two kidney, one clip" (2K1C) renovascular hypertension model in rats. The studies were carried out on the stomach of rats. After 6 week period of the renal artery clipping procedure, eight 2K1C rats developed stable hypertension. The hypertension significantly increased the number of endocrine cells pylorus immunoreactive to calcitonin gene-related peptide (CGRP) antisera. The differences between the hypertensive rats and the control group concerned not only the number of endocrine cells but also their distribution. CGRP participates in the regulation of cardiovascular functions both in normal state and in the pathophysiology of hypertension through interactions with the prohypertensive systems. The changes induced by hypertension in the neuroendocrine cells containing CGRP of the rats are discussed.

Plasmin system regulation in an ovalbumin-induced rat model of asthma.

BACKGROUND: So far studies showing the role of the plasmin system in airway remodelling have been conducted using in vitro models. The aim of the present study was to determine plasmin system regulation in an in vivo rat model of asthma. METHODS: Asthma in Wistar rats was induced by ovalbumin (OVA) sensitization followed by an OVA challenge (OVA/OVA, n = 6). Control groups were saline-sensitized challenged with OVA (VEH/OVA, n = 6) and OVA-sensitized challenged with saline (OVA/VEH, n = 6). Plasmin system components were determined in the plasma by ELISA. Plasminogen activator inhibitor-1 (PAI-1) was localized by an immunohistochemical reaction. RESULTS: Sensitization and challenge with OVA caused thickening of the airway wall, hypertrophy of smooth muscle cells, infiltration of inflammatory cells, subepithelial fibrosis, epithelial and endothelial lesions. Serum total IgE was significantly higher in OVA-sensitized rats as compared to VEH-sensitized control groups. Tissue plasminogen activator activity was significantly decreased in asthmatic animals (4.48 +/- 0.4 vs. 6.7 +/- 0.3 ng/ml for OVA/OVA and OVA/VEH; p < 0.05), and PAI-1 activity was statistically significantly higher in asthma rats (0.8 +/- 0.05 vs. 0.5 +/- 0.03 ng/ml for OVA/OVA vs. OVA/VEH; p < 0.05). alpha2-Antiplasmin was higher in rats receiving OVA sensitization than in those that were sham sensitized (p < 0.05). Immunohistochemical staining for PAI-1in the lungs of asthmatic animals showed very strong PAI-1 expression in lung inflammatory cells. CONCLUSIONS: We have demonstrated for the first time the existence of PAI-1 in lung inflammatory cells of rats with asthma. This finding was consistent with the superiority of plasmin system inhibition over activation in plasma.

Impression cytology of the conjunctival epithelial cells in patients with cystic fibrosis.

PURPOSE: Cystic fibrosis (CF) is the most frequent lethal autosomal recessive hereditary disorder. The disease affects all secretory epithelia, including the eye, and belongs to the group of ocular surface epithelial diseases, termed keratoconjunctivitis sicca or dry eye syndrome. The aim of this study was to evaluate goblet cell population and conjunctival epithelial morphology in patients with CF. METHODS: A total of 20 CF patients and 20 controls underwent conjunctival impression cytology. RESULTS: Impression cytology showed conjunctival squamous metaplasia and goblet cell loss in patients with CF. CONCLUSION: Reduced goblet cell numbers and squamous metaplasia may be indicative of a higher degree of epithelial damage of conjunctival epithelial cells in CF patients, and the presence of neutrophils is a strong sign for an inflammatory background of this disease.In view of the simple, noninvasive nature of impression cytology, this technique may prove to be an important tool for the diagnosis and monitoring of dry eye changes in CF patients.

An immunohistochemical study of endocrine cells in the stomach of hypertensive rats.

Essential hypertension is a complex disease with both genetic and environmental determinants. The effect of spontaneous hypertension on the distribution and occurrence of somatostatin-, gastrin- and serotonin-immunoreactive cells in the fundus and pylorus of the rat stomach was examined by immunohistochemistry. The animals were killed by decapitation at 4 and 16 weeks of age (5 control rats and 5 hypertensive rats). Endocrine cells generally increase in number in hypertensive rats as compared to control rats. However, the detailed responses of endocrine cells to hypertension depend on the cell type, region of gastric mucosa and age of animals. The present results suggest that hypertension has an influence on the intrinsic regulatory system by endocrine cells control in the rat stomach.

Calcitonin gene-related peptide expression altered in pulmonary neuroendocrine cells in an experimental model of uraemia.

The undertaken studies is aimed at immunohistochemical localisation, quantitative assessment and functional evaluation of neuroendocrine cells in the lungs of rats with experimentally induced uraemia. Lung and trachea fragments were collected after 1, 2 and 4 weeks from nephrectomy. Paraffin-embedded sections were stained with haematoxylin and eosin and by silver impregnation. An immunohistochemical reaction was then performed with the use of a specific antibody against calcitonin-gene related peptide (CGRP) to identify neuroendocrine cells. Obtained results of the performed studies demonstrated a significantly increased number of CGRP-immunopositive cells in the lungs of applied uraemic rats (4.47+/-0.97, 7.62+/-1.61 and 5.72+/-2.5 neuroendocrine cells/mm(2) of lung section after the 1(st), the 2(nd) and the 4(th) week, respectively), when compared with that in the control (1.22+/-0.47 neuroendocrine cells/mm(2) of lung section). The obtained results may be approached as a morphological expression of neuroendocrine cells hyperfunction in the lungs in result of disturbed internal body environment caused by renal parenchyma impairment. The enhanced activity of neuroendocrine cells, observed in the lungs of uraemic rats, was confirmed by results of studies of morphometric parameters, such as: area, diameter, length, width and the circularity index.